Monday, February 10, 2014

PCR will find it...

Commissioned by the Alpaca TB Support group, the study was seeking a more accurate test for screening alpacas and llamas for zTuberculosis. And as some fairly hefty sums were bandied about regarding the cost of collecting suitable ante mortem samples, it was suggested that as AHVLA had no shortage of alpaca carcases coming through their doors, that these could provide the relevant material to kick start the project.

There is a difference between how the screening tests in use for zTuberculosis work.
The intradermal bovine skin test seeks an immune response from the candidate, to exposure to the bacteria which may go on to cause zTB. Various blood assays seek antibodies which the candidate may have thrown up to fight infection from z TB but PCR (Polymerase Chain Reaction) seeks the bacteria itself.
The skin test has proved extremely inconsistent when used on alpacas. And by that we mean, it's rubbish.

Blood assays, as applied to camelids in a confirmed TB breakdown herd, can
throw up some false positives (3-4%), which is upsetting for the owners. Thus a different ante mortem test was sought - with dead alpacas, already in the AHVLA system providing the raw material.

The first tranche of samples were taken from animals which at post mortem showed advanced gross pathology. (Large and extensive lesions) The Interim report into this part of the study, we explained in this posting. - [link] And as expected, the more m.bovis bacteria available in the sample, the better the result using PCR. Disease had already been confirmed in the animals used in this project by postmortem, gross pathology and cultures.

A second tranche of samples were taken from animals with much less advanced disease and some may have been from lesions which were not shedding bacteria into the locations from which the two PCR samples (sputum and faeces) were collected.
Nevertheless, with no false positives, if m.bovis was present in the samples offered, PCR appeared to be able to find it.

A combination of sputum and faecal samples gave even better results. The paper's authors suggest pooled samples (as with m.aviumparatuberculosis) could be offered as a herd screen and they describe as 'highly significant' the increasing trend of positive PCR results in line with an increasing pathology score.

Their conclusions include:

"While more SACs (South American Camelids) were positive in the faecal PCR, problems associated with collection of the nasal swabs post mortem may have compromised the test on this sample type. A combination of the nasal swab PCR and faecal PCR appeared to have a marginally greater sensitivity than either of the tests alone.

Testing of both sample types may increase sensitivity but at increased cost. Nasal swabs and faeces samples are relatively easy for owners to collect though there needs to be consideration of the possible exposure of owners to M. bovis when collecting nasal swabs.

Pooling of samples from multiple SACs may potentially be a useful
screening test.

There is a highly significant increasing trend in the proportion of positive results in the nasal swab and faecal PCR tests with increasing pathology score. This confirms, as expected, that a PCR test on clinical samples will have greatest sensitivity in SACs with more extensive pathology.

The number of SACs where culture results were available was small but the results were in agreement with the PCR results apart from three which were culture positive and PCR negative. This finding is not unexpected as post mortem and culture will be more sensitive than a PCR test on clinic samples."

This is a huge leap forward in validating this technology for use ante mortem in alpacas, but also in other species -[link] where gross pathology indicates a huge bacterial spread.